γ-trifluoromethyl proline: Evaluation as a structural substitute of proline for solid state 19 F-NMR peptide studies

Transcription

1 Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry. This journal is The Royal Society of Chemistry 2015 γ-trifluoromethyl proline: Evaluation as a structural substitute of proline for solid state 19 F-MR peptide studies Vladimir Kubyshkin, Sergii Afonin, Sezgin Kara, ediljko Budisa, Pavel K. Mykhailiuk and Anne S. Ulrich Contents Copies of the MR spectra for the compounds Thermodynamic parameters of the trans-cis rotation in 8... Kinetic parameters of the trans-cis rotation in 8 Solution MR spectra of the peptides Supporting solid state MR spectra pk a determination for the amino group of TfmPro S2 S4 S6 S8 S9 S15 S17 S21 S24 S26 S1

2 Copies of the MR spectra for the compounds 2-tert-butyl 2-methyl (2S)-4-trifluoromethyl-3-pyrrolin-1,2-dicarboxylate (6) Boc CO 2 Me 1H ppm Boc CO 2 Me J = 20 Hz 19F ppm S2

3 Boc CO 2 Me dept135 dept90 13C CPD ppm S3

4 Methyl (2S,4S)--tert-buthoxycarbonyl-4-trifluoromethylprolinate (7) Boc CO 2 Me 1H ppm Boc CO 2 Me J = 8 Hz 19F ppm S4

5 Boc CO 2 Me dept135 dept90 13C CPD ppm S5

6 (2S,4S)--(9-fluorenylmethoxycarbonyl)-4-trifluoromethylproline (9) CO 2 H Fmoc 1H ppm CO 2 H Fmoc 19F ppm S6

7 CO 2 H Fmoc dept135 dept90 13C CPD ppm S7

8 (2S,4S)--(tert-butoxycarbonyl)-4-trifluoromethylproline (1) Boc CO 2 H 1H ppm S8

9 Methyl (2S,4S)--acetyl-4-trifluoromethylprolinate (8) ppm S CO 2 Me Ac 1H 700 MHz s-trans s-cis ppm

10 DMSO-d6 Ac CO 2 Me 1H, 500 Hz t, 8.3 Hz CDCl3 t, 8.3 Hz D2O t, 8.3 Hz ppm S10

11 DMSO-d6 CO 2 Me Ac 19F, 470 MHz 304 K ppm ppm CDCl3 D2O S11

12 (only the major rotamer labelled) dept135 CO 2 Me Ac dept90 dept45 13C CPD, 176 MHz ppm S12

13 1H J-resolved, 700 MHz Current Data Parameters AME wova_pro EXPO 27 PROCO 1 Hz -20 Ac CO 2 Me F2 - Acquisition Parameters Date_ Time ISTRUM spect PROBHD 5 mm PABBO BB- PULPROG jresqf TD 4096 SOLVET D2O S 4 DS 16 SWH Hz FIDRES Hz AQ sec RG DW usec DE usec TE K D sec D sec I sec -15 ======== CHAEL f1 ======== SFO MHz UC1 1H P usec P usec PLW W -10 F1 - Acquisition parameters TD 64 SFO MHz FIDRES Hz SW ppm FnMODE QF F2 - Processing parameters SI 2048 SF MHz WDW SIE SSB 0 LB 0 Hz GB 0 PC F1 - Processing parameters SI 256 MC2 QF SF MHz WDW SIE SSB 0 LB 0 Hz GB ppm S13

14 1H13C HSQC, 700/176 MHz Current Data Parameters AME wova_pro EXPO 26 PROCO 1 Ac CO 2 Me ppm F2 - Acquisition Parameters Date_ Time ISTRUM spect PROBHD 5 mm PABBO BB- PULPROG hsqcetgpsi2 TD 2048 SOLVET D2O S 2 DS 16 SWH Hz FIDRES Hz AQ sec RG 2050 DW usec DE usec TE K CST D sec D sec D sec D sec D sec D sec I sec ZGOPTS 30 ======== CHAEL f1 ======== SFO MHz UC1 1H P usec P usec P usec PLW W ======== CHAEL f2 ======== SFO MHz UC2 13C CPDPRG[2 garp P usec P usec PCPD usec PLW W PLW W ====== GRADIET CHAEL ===== GPAM[1] SMSQ GPAM[2] SMSQ GPAM[3] SMSQ GPAM[4] SMSQ GPZ % GPZ % GPZ % GPZ % P usec P usec ppm F1 - Acquisition parameters TD 256 SFO MHz FIDRES Hz SW ppm FnMODE Echo-Antiecho F2 - Processing parameters SI 1024 SF MHz WDW QSIE SSB 2 LB 0 Hz GB 0 PC F1 - Processing parameters SI 1024 MC2 echo-antiecho SF MHz WDW QSIE SSB 2 LB 0 Hz GB 0 S14

15 Thermodynamic parameters of the trans-cis rotation in 8 Basic experimental procedure is described in the main text. 19 F MR 30-deg flip angle spectra were collected on 471 MHz resonance frequency without or with 1 H decoupling (inverse gated, no OE enhancement). The 1D time domain contained data-points making 2.8 s acquisition time. Recycling delay was 1 s. The spectra were collected from 4 steady state and 32 recorded transients. Time domain spectra were processed with Gaussian windowing (gb = 0.05, lb = -1 Hz) in a phase sensitive fashion, baseline corrected and integrated. The K values were calculated directly from the integrals and converted into G for plotting. 19F{1H} incorrected chemical shifts 90.0 C 78.6 C 67.2 C 55.8 C 44.4 C 29.6 C ppm - measured in deuterium oxide. The shift due to the lock (water) resonance temperature shift was not corrected. S15

16 T calibrated, K T, C K tc G, kj/mol linearization: G, kj/mol y = Intercept + Equation B1*x^1 Weight o Weighting Residual Sum of Squares Adj. R-Square Value Standard Error Intercept dg B E T, K Determined parameters of the rotation: H = -4.85±0.11 kj/mol S = -4.72±0.33 J/mol K S16

17 Kinetic parameters of the trans-cis rotation in 8 Basic experimental procedure is described in the main text. The 19 F EXSY spectra were collected on 471 MHz resonance frequency without decoupling. The spectral width was 2.0x2.0 ppm, time domain array 2048x512 data-points, making acquisition times of 1.11x0.26 s. Recycling delay was 1 s, number of scans 2. The spectra were processed with sinc squared windowing on both dimensions in a phase sensitive mode, baseline corrected and integrated. 128 data-points were linearly predicted in the indirect dimension. The difference to a standard OESY processing setup was the use of squared sinc function instead of conventional squared sine bell. This change was done in order to make additional smoothening of the peaks for integration and reduce truncation noise. Exchange rate matrices calculations were performed in EXSYCalc (Mestrec), where EXSY integral values were inset. Secondary diagonal elements were the k values given in the table below: S17

18 T calibrated, K T, C k 1, s -1 k -1, s S18

19 Eyring plots: for the k y = Intercept + B Equation 1*x^1 Weight o Weighting Residual Sum E-4 of Squares Adj. R-Square Value Standard Error ln(kct/t) Intercept ln(kct/t) B ln(k ct /T) /T, K -1 for the k y = Intercept + B Equation 1*x^1 Weight o Weighting Residual Sum of Squares Adj. R-Square Value Standard Error ln(ktc/t) Intercept ln(ktc/t) B ln(k tc /T) /T, K -1 S19

20 Determined parameters of the process: for k 1 : H = 78.3±0.3 kj/mol S = -11.9±1.0 J/mol K For 300 K E a = 81.9±0.6 kj/mol for k -1 : H = 81.4±0.5 kj/mol S = -12.6±1.6 J/mol K For 300 K E a = 85.2±1.0 kj/mol E a (cis/trans trans/cis)= -3.4 kj/mol S20

21 Solution MR spectra of the peptides 19 F MR spectra of 2TfmPro-GS in different media (at 298 K): 19F W5 (centered ppm) in PB-TFE 19F in PB / SDS micelles 19F in DMSO-d ppm S21

22 red - GS (wild type) blue - 2TfmPro-GS 1H TOCSY (with water pre-saturation) in DMSO ppm ppm S22

23 red - GS (wild type) blue - 2TfmPro-GS 1H TOCSY (H2O W5, TFE pre-saturated) in PB-TFE ppm ppm S23

24 Supporting solid state MR spectra 31 P MR of the 2TfmPro-GS/DLPC oriented sample before and after the 19 F MR temperature series reported in the main text. The spectra were measured well above the phase transition of DLPC at 303 K. Sample macroscopic placement was such that the membrane normal was parallel to the magnetic field. The percentage of the desired macroscopic alignment was found to be higher than 80 %. after ppm before 31P S24

25 19 F MR powder static and magic angle spinning (MAS) spectra of 2TfmPro-GS for estimation of the maximal anisotropic parameters. The spectra were taken at 298 K and MHz. The isotropic chemical shift was found to be ppm. S25

26 pk a determination for the amino group of TfmPro 25 mg of 1 was dissolved in 1 ml dichloromethane and 0.5 ml trifluoroacetic was added. The solution was stirred at the room temperature for an hour and the volatiles were blown off by excessive nitrogen current. The rest was dissolved in 50 µl of water and resulting solution was aliquotized 2µl in the MR tubes containing 550 µl of 150 mm sodium phosphate buffer. The final concentration of TfmPro in the buffered systems was therefore ~ 6 mm. The last datapoint at ph 13 was measured in 0.1 M aoh. 19 F MR spectra were measured in inverse gated decoupled 30-degree flip angle experiments at 471 MHz frequency and 298 K. The residual TFA signal was observed at ±0.01 ppm in all samples and the ratio between TfmPro and TFA was found as 1.3. From these measurements several points deviated from the sigmoidal behavior in the range In this range the buffer capacity of phosphate buffer is too low to quench 16 mm acidic additive. Therefore we collected additional dataset with diluted TfmPro*TFA. Such that the final concentration was 0.6 and 0.16 mm. All datapoints were collected together except of the ones rejected where preference was given to the more diluted samples. The found 19 F chemical shifts of TfmPro were plotted against ph as depicted, fitted according to the Boltzman fit (OrifinPro 9.1, alternatively logistic fit with similar outcome) and the first derivative of the fit delivered the maximum point 8.5. Apparent standard error was 0.1 (electrode). 31 P MR chemical shifts were used for control. Two series showed after all a good consistency to one another. The bending point for the second ionization constant was found at 6.8 in both series. δ( 19 F) TfmPro, ppm d(δ fit)/dph δ( 31 P), ppm data points Boltzman fit rejected points ph TfmPro*TFA ~ 6 / 10 mm TfmPro*TFA ~ 0.6 / 1 mm ph ph = ph S26